Project Summary/Abstract Sudden unexpected death in epilepsy (SUDEP) remains a major concern for persons with epilepsy and their families. The incidence of SUDEP is estimated at 1 to 2.5 per 1000 patient years however the underlying reasons are poorly understood. Here we will address the adenosine hypothesis of SUDEP. Epileptic seizures trigger a surge of adenosine (ADO), which is an endogenous agent required for seizure termination, but which also controls respiratory functions in the brainstem by activation of different subtypes of adenosine receptors (ARs). Our overarching hypothesis is that a seizure-induced increase in ADO in combination with insufficient metabolic clearance of ADO in the brainstem can cause fatal over-activation of ARs leading to brainstem dysregulation and respiratory dysfunction as a precipitator of SUDEP. In support of our hypothesis, we demonstrated that a non-selective AR antagonist (caffeine) reduced lethal apnea in rodent models of epilepsy and traumatic brain injury. Our preliminary data support a strong association between ADO metabolism, AR activation, and lethal apnea. For those reasons we focus our proposal on the brainstem- dependent respiratory mechanisms of SUDEP. Specifically, we will address our central hypothesis that the susceptibility to SUDEP is related to abnormal ADO metabolism and signaling in respiratory regions of the brainstem and preventable by therapeutic interventions that enhance metabolic clearance of ADO or block AR activation. Brainstem specific manipulations will allow us to test a specific role of ADO in the brainstem. Dissection of seizure-related functions of ADO metabolism and signaling in the limbic system from respiratory control functions in the brainstem will enable a translational path for ADO-manipulation for the prevention of SUDEP. Our model system is a `SUDEP-prone mouse' with impaired metabolic clearance of ADO due to a heterozygous disruption of the adenosine kinase (Adk) gene. ADK, expressed in astrocytes is the main metabolic clearance route for ADO in the brain. Our research goals will be approached in 3 Specific Aims: (1) Test hypothesis that SUDEP is associated with maladaptive changes in ADO metabolism. (2) Test hypothesis that caffeine plays a critical role in SUDEP susceptibility. (3) Test whether suppression of A2AR activation can prevent SUDEP. The expected outcome of this research is the demonstration that the capacity for the metabolic clearance of seizure-induced ADO through the brainstem determines susceptibility to SUDEP. Mechanistic studies will identify the AR subtypes involved. In addition, this research will help to clarify whether the chronic use of caffeine is of benefit or presents a risk factor for persons with epilepsy. Finally, we will test whether A2AR antagonists, which are already in clinical development for the treatment of Parkinson's disease, can prevent SUDEP.